Alpco diagnostics are a supplier of immunoassay kits for the medical research community.
Their kits are developed by scientists committed to supporting those who work in physiology and disease research.

ALPCO provide a broad portfolio of life science research tools for academic, pre-clinical and clinical use, including primary antibodies, flow cytometry reagents, recombinant proteins, and products for HPLC and LC-MS/MS, but are best known for providing, as they put it: “Immunoassays Beyond the Ordinary.”

ALPCO have a reputation for quality among their customers because they focus on providing immunoassays which are highly sensitive, specific and reproducible with a broad dynamic range that allows for fewer dilution steps and therefore reduced systematic error.

They have recently developed the new STELLUX™ range of chemiluminescent immunosorbent assays. These combine exquisite sensitivity, ease of use, and outstanding reliability which comes from extensive testing and characterisation.


Product Categories

Applications

Immunoassays

An immunoassay is a method used by many different fields of science to determine the presence of an analyte in a sample. The method is based on the interaction between the antibodies and the analyte, and the results can be reported in concentrations, positive/negative or greater than/less than thresholds.

Immunoassays are used in academic, pre-clinical and clinical research, clinical diagnosis and in supporting technologies in many different fields of science such as pharmaceuticals, veterinary, forensic, military and food sciences.

Commercially available assays are designed and optimized for specific analytes, sample types, sample volumes and ranges. The protocol contained with the product should be strictly followed, without deviation, to obtain the most accurate and reproducible results.

Immunoassays report results in either a qualitative or quantitative manner. Qualitative results are reported as positive/negative or greater than/less than thresholds, while quantitative results are calculated concentrations. Assay results are achieved through either a direct or competitive interaction.

Direct Assay

The analyte present in the sample is measured directly using two antibodies for capture and detection. The analyte will be captured by the antibody adhered to the plate and detected using a second antibody conjugated to a detection molecule. The detection molecule will display a signal that can be measured. The level of signal produced and the analyte concentration are proportional.

Competitive Assay

The analyte present in the sample is measured directly using two antibodies for capture and detection. The analyte will be captured by the antibody adhered to the plate and detected using a second antibody conjugated to a detection molecule. The detection molecule will display a signal that can be measured. The level of signal produced and the analyte concentration are proportional.

Antibodies

Assays use the kinetics of the antibody:antigen interaction to generate a standard curve using known values to interpolate unknown values.

Standards/Calibrators

Standards/Calibrators are a set of known concentrations from which a curve can be generated. They contain specific concentrations of analyte in a matrix similar to the sample type recommended for the assay. The signal values obtained from the analyte:antibody interactions in the standards allow an algorithm for a curve to be calculated. The sample results are interpolated based on this algorithm as the interactions between the analyte in the sample and the antibodies should relate to the interactions in the standards.

Substrates

  • Enzymatic: an enzyme is conjugated (chemically attached) to the antibody. A substrate is necessary to bind with the enzyme and produce the signal.
  • Colorimetric: the enzyme converts a substrate to produce a colored reaction product.
  • Fluorescent: the enzyme converts a substrate to a reaction product that fluoresces when excited by light.
  • Luminescent: the enzyme converts the substrate to a reaction product that emits photons of light.
  • Radioactive: radio isotopic tracers are conjugated to the antibody. Gamma or beta scintillation detection equipment is used to detect the radioactivity in Counts Per Minute (CPM).

Can I read the plate without having a reference wavelength?

A. Yes, you can. However, the use of a reference wavelength may improve the sensitivity of the assay.

What kind of sample type can be used with the kit I purchased?

A. Each type of kit has been validated for the sample types indicated in its protocol. All other sample types would have to be validated internally. Please contact our technical support team for further information regarding possible testing of other sample types performed by the product users.

Is this kit cross reactive with another species?

A. Each type of kit has been validated for the species indicated in the protocol. Although cross reactivity may exist, validation of other species would need to be performed internally. Please contact our technical support team for further information regarding possible species validation performed by the product users.

How many standards and controls do I run?

A. Each kit will include at least one set of standards (or one vial of standard to be diluted) and in most cases one or two controls. Standards and controls (if included) must be run each time the assay is performed and it is highly recommended to run all standards/controls/and samples in duplicate.

Can I use reagents from other kits or lots?

A. No. Do not substitute components from other kits or lots.

Can I modify incubation times or temperatures?

A. No. Please do not deviate from the protocol.

Can I use an expired kit?

A. No. The kit should not be used if it is past the expiration date specified on the kit label.

Can I use a kit that has been left at room temperature?

A. Please be sure that the kit and its components are stored as indicated in the kit insert. For additional information about kit stability at room temperature please contact our technical support team.

Can I skip running the control(s) on my assay?

A. No. If a control(s) is provided always be sure to run the control(s) with each assay. The concentrations of the controls should fall within the range specified on the certificate of analysis.

Is it necessary to make a layout of my samples?

A. It is extremely important to know where your standards, controls and samples are located on the plate so that results are generated appropriately. Click here for a downloadable platemap. It also helps to label the strips on the plate (use small tabs at each end) in the event that strips become loose while decanting.

Why is the washing step so important?

A. Correct washing of the plate is a very critical and important aspect of running any successful ELISA. Consistency in washing the plate is essential. Washing the plate too rapidly or too slowly, incomplete washing or aspirating, and allowing the wells to sit dry are all factors that will affect the precision of the assay. We do not recommend the use of a multichannel pipette for this purpose. Multichannel pipettes do not apply enough pressure to thoroughly remove the unbound material and this will result in higher background noise. A video displaying proper washing technique is available in the Learning Center section of the website or click here.

How many times can I freeze and thaw my samples?

A. Recommended freeze/thaw cycles vary based on kit. We recommend to avoid repeated freeze and thaw cycles unless otherwise indicated in the manual. If the collected sample amount allows for it, prepare and freeze aliquots to eliminate or reduce freeze/thaw cycles.

Can I use my own "homemade" buffers?

A. Each kit contains diluents and buffers that are formulated to closely match specific sample types. The manufacturer will not guarantee the performance of the kit if components that were not provided with the kit are used to run the assay.

What should I do if I run out of diluent?

A. Enough reagents are provided to run the entire kit when the protocol is followed. If your type of study requires a greater dilution, calculate the amount of diluent needed prior to setting up the assay and additional diluent may be available for purchase. Please contact customer support for information on the availability of individual components.

My controls are out of range. Is my data invalid?

A. The concentration of the controls must be within the range specified in the certificate of analysis. If the controls are out of range the assay may be invalid. Also, make sure the curve fit recommended in the protocol has been used. If more than one recommendation is provided use the regression method that best fits the standard data points.

I didn't get good values from my standard curve. Can I use the example values from the protocol?

A. No. Never use example values to calculate your assay results. Sample concentrations should be generated from the standard/calibrator values obtained with each assay.

My samples generated OD values out of the standard range. Can I still calculate concentrations?

A. Only sample values that fall within the range of the standard curve can be used. Values outside of this range are generally not linear and can lead to incorrectly extrapolated results.

My standard curve is fine but I didn’t get a signal with my samples. Why?

A. The sample may contain the analyte but it is undetectable based on sensitivity of the kit. The matrix of the sample may also be masking the detection. Ensure that the dilutions have been made as stated in protocol and review the procedure to ensure all reagents were added with the correct volume and in the correct order.

How many samples can I run?

A. ALPCO now offers a tool to help with calculating the number of samples that can be run in a kit. Once you review the protocol to determine the number of standards, controls, and blanks that may be required for the specific kit you plan to run click here to go to the Sample Calculator.

What is reference wavelength?

A. A reference wavelength is a secondary wavelength used for correction (normalization) of the principal wavelength OD values. It helps to account for imperfections in the plate.

Why is 450nm the most common measure wavelength?

A. The measurement wavelength is determined by the substrate used in each kit. The most common one is TMB. It produces a blue color measurable at a wavelength of 650nm. It can be used in end point assays by stopping the reaction with either 1M phosphoric acid or 1M sulfuric acid. A yellow reaction product is formed upon acidification that is measurable at 450 nm.

What is the difference between measurement wavelength and reference wavelength?

A. The main difference between measure wavelength and reference is that the first one has been chosen to read the absorbance produced by the substrate and the second one (reference) to detect imperfections in the plate.

Poor precision between replicates for standards and samples (high CV values)

Inadequate plate washing

Problem with multichannel pipette during the addition of the conjugate or substrate

Pipetting technique

Bubbles in wells

Poor precision between replicates for samples only (high CV values)

Inadequate sample mixing

Pipetting technique

Inadequate plate washing

Carry over from high and low samples

Wrong sample dilution buffer

Poor standard curve (does not match previous results or Certificate of Analysis)

Inadequate plate washing

Pipetting technique

Inadequate mixing of reagents

Insufficient shaking of plate

Wrong volume of reagents added to the well

Storing of prepared reagents

Low or inconsistent ambient temperature

Differences in chemiluminescence instrumentation

All wells turned bright blue for colorimetric assay

Inadequate plate washing

Contamination of the substrate with enzyme conjugate

High background (blank or 0 standard values are too high)

Inadequate plate washing

Old or contaminated wash buffer

Contamination of the substrate with enzyme conjugate

Wrong conjugate dilution

Wrong filter used in the plate reader

No color development or very low OD readings for standards and samples

Wrong filter used in the plate reader

Error in reagent preparation

Inadequate mixing of reagents

Insufficient shaking of plate

Mix-up in reagents

Reagents contaminated or added in the wrong order

Alternate component lot or expired component used

Assay incubation times not followed correctly

Delayed reading of plate

No color development or very low OD readings for samples only

Error in experimental design

Incorrect sample handling

Error in sample dilution calculations

Non-validated sample type

Insufficient shaking of plate

Control out of range

Incorrect reagent handling

Poor standard curve

Error in reconstitution of standards and/or controls

Wrong curve fit used

Unexpected results for samples

Improperly prepared sample

Sample IDs mixed up

Error in sample dilution

Discrepancy in reported units

Error in experimental design

Interfering substances

Specificity

Assay drift: difference in values from beginning to end of plate

Delays due to reconstitution or dilution

Reagents not at temperature

Extended time between addition of reagent to first well and last well

Inter-assay variability (poor precision for controls or samples tested on different plates)

Inter-operator variability

Differences in pipette calibration

Differences in instrumentation

Variation in environmental conditions

Differences in sample handling

Tools

Plate Shaking Techniques
Instructions for proper plate shaking with an ELISA assay
Sample Calculator
“How many samples can I test on my 96 well ELISA plate?

Videos

Plate Washing Technique Video
Recommendations for hand washing and drying an ELISA plate

Chemiluminescence

Chemiluminescence is the emission of light (luminescence), or heat as the result of a chemical reaction. STELLUX® assays utilize GLOW chemiluminescence using enzymes and substrates that generate light from an enzyme mediated chemical reaction. Several different chemical reactants/reactions produce luminescence (1,2-dioxetanes, luminols, acridinium esters, etc.).

Glow vs. Flash Chemiluminescence:

  • Intensity/Time profile consists of an initial rise period and prolong emission
  • Provides greater flexibility in measurement process (standard ELISA plate readers)
  • Light intensity at any time point through the plateau can be related to the amount of analyte
  • Biotin/Streptavidin reaction dramatically increases sensitivity

The platform features include:

  • Broad dynamic range
  • Superior sensitivity, dilutional linearity, precision and accuracy
  • Smaller sample volumes
  • Less background
  • Performance data, including lot validation package

Chemiluminescent Plate Reader Settings

Reader Settings

HPLC/LC-MS

HPLC stands for High-Performance Liquid Chromatography. It uses the chemical properties of a stationary phase (the column) and a mobile phase (the buffer) to separate complex mixtures of molecules. The column serves to slow the movement of compounds to varying degrees based on their chemical or physical properties.

By varying the properties of the two phases and the runtime parameters, different compounds can be separated in different ways. The column parameters can vary based on particle or pore size, internal diameter, column length and flow rate through the column. The buffer can vary based on composition (differing levels of aqueous or organic solvents) or by changing the solvent concentration over time (isocratic versus gradient).

Samples may have to be prepared in special buffers prior to loading on the machine. Any particulate in the sample has the potential to clog the column, so it is preferable to separate prior to running. Often there is an in-line Solid Phase Extraction column to remove what is not soluble in the sample buffer.

After being separated by HPLC, each compound is detected by UV/Vis and represented in a chromatogram where the y-axis is absorbance intensity (compound amount) and the x-axis is time (how long the compound was retained). In some cases, compounds will be further analyzed by Mass-Spectrometry.

Size-exclusion

Size-exclusion separates based on compound size. The column consists of trillions of microscopic pools of solvent around the beads. Smaller proteins will be caught in these pools while larger proteins pass through and elute out faster. The longer the time of elution, the smaller the protein collected.


Reverse-Phase

Reverse-Phase separates based on compound polarity. The column is a non-polar material, often silica based, and the mobile phase is a varying amount of aqueous (polar) and organic (non-polar) solvents, often with a highly polar ion added in. The longer the elution time, the higher the over-all polarity.


Ion-exchange

Ion-exchange separates based on compound charge. In this method, the mobile phase contains chemicals that induce charge on the compound of interest. This can be done by cations, anions, pH or temperature.


Affinity columns

Affinity columns separate based on the formation of stable, specific and reversible complexes. This can be done using antibodies, receptors or their respective ligands. By lining the column with antibody, the analyte of interest will be bound and isolated. Later, the mobile phase can be changed so the antibody no longer binds, and the analyte is then eluted and collected.

What are the basic components of an HPLC system?

A. A functioning HPLC system includes a sampler, pump, column, detector and data processor. A degasser and column oven may also be used.

How many types of detectors are available for HPLC?

A. There are UV, fluorescence, electrochemical, conductivity, refractive index, evaporative light scattering, chiral, radioactive and mass spectrometry detectors.

I am not sure which column to use?

A. Check the assay protocol to instructions on the appropriate column. The most commonly used column is C18 which covers a wide range of applications.

How do I make a column last longer?

A. Filter samples to remove particulate and make sure the pH of the mobile phase is within specifications for the column. If the column will be stored for an extended period of time, flush with methanol or acetonitrile.
No peaks
Double peaks
Contaminating peaks
Variable retention times
Baseline is drifting
Baseline is not smooth

Therapeutic Areas

Our extensive selection of antibodies is validated for use in flow cytometry, immunohistochemistry and other applications, and allows researchers to identify hundreds of intracellular and extracellular proteins.

Antibodies are produced by the immune system in response to antigens. Though the general structure of antibodies is very similar, a small variation in the antigen-binding site of the protein allows for the specific recognition of a single antigen.

Our reliable testing solutions allow for the treatment, control and prevention of conditions associated with allergies and infectious disease.

The immune system protects our body against several factors including allergens, harmful pathogens and foreign particles. Allergies involve an exaggerated response of the immune system and are among the most common chronic conditions worldwide. Infectious disease agents are also one of the leading contributors to debilitating and sometimes fatal conditions. Therefore, scientists look to understand interactions between microbes and the immune system.

We strive to help scientists better understand the relationship between key regulators of bone metabolism.

Bone metabolism is a continual, cyclic interplay of bone growth and resorption. The dynamic relationship between osteoclasts, osteoblasts and an array of hormonal and regulatory influences carefully orchestrates this process. The relative levels of these signaling molecules dictate whether healthy, balanced bone metabolism ensues. Disturbances to this delicate equilibrium, where reabsorption is greater than growth, can weaken the skeletal architecture and put one at risk for the development of chronic and debilitating diseases, such as osteoporosis.

We offer a range of research tools required to identify novel Cardiovascular Disease and Oxidative Health biomarkers, enabling scientists to test their relevance in basic, preclinical and clinical studies.

Cardiovascular Disease (CVD) encompasses a constellation of diseases related to the heart and/or circulatory system. Two primary risk factors for CVD include obesity and Type 2 Diabetes, both of which have continued to be on the rise over the past few decades. It is evident that regardless of the advancements in scientific knowledge of the disease and the improvements in diagnosis and treatment, more research is necessary to further our understanding of CVD.

As a leading provider of testing solutions for the Diabetes and Obesity research community, we support scientists’ efforts to gain a greater understanding of these conditions.

With no cure for Type 1 or Type 2 Diabetes and a lack of effective obesity treatments, scientists in the Diabetes and Obesity fields are continually in need of new methods to advance their studies. As these needs progress, we strive to provide Diabetes and Obesity investigators with the necessary tools to identify key biomarkers to achieve their research goals.

We offer advanced tools to study gastroenterology and its related fields, including areas such as food intolerance and differentiating between IBD and IBS.

Gastroenterology is the branch of medicine that deals with the study and treatment of the digestive system (comprised of stomach and intestine) and related disorders affecting it. Gastric diseases and ailments have become a growing therapeutic segment of interest due to an increased prevalence of irregular dietary habits and poor hygiene factors across the world.

We offer advanced tools to study gastroenterology and its related fields, including areas such as food intolerance and differentiating between IBD and IBS.

Gastroenterology is the branch of medicine that deals with the study and treatment of the digestive system (comprised of stomach and intestine) and related disorders affecting it. Gastric diseases and ailments have become a growing therapeutic segment of interest due to an increased prevalence of irregular dietary habits and poor hygiene factors across the world.

We supply immunology researchers with a wide array of solutions to gain insight into the development and progression of immune responses.

Immunology is the branch of biomedical science that is concerned with the relationship between body systems, pathogens and immunity. The central science of immunology is the study of the molecular and cellular components that comprise the immune system, including their function and interaction.

We offer scientists a number of relevant assays in various formats for the investigation of neurological function, diseases and disorders of sensory information processing.

Neurological disorders can be caused by a variety of systematic diseases, toxic exposures, medications, infections and hereditary disorders.(1) Studies have linked the presence of high anti-ganglioside titers to several neuropathies, Additionally, correlations have been drawn between the severity of the disorder and the isotype detected. IgG antibodies have been strongly associated with acute conditions, while IgM antibodies tend to be present in chronic diseases. Many of these neurological disorders exhibit similar symptoms involving sensory loss and distal pain, numbness, tingling and weakness, causing them to be difficult to dissociate. An increasing level of research indicates immunological tests for ganglioside antibodies plays an important role in the diagnosis and management of neuromuscular diseases.(2)

1. Azhary, Hend. et al. Peripheral Neuopathy: Differential Diagnosis and Management. American Family Physician. Apr 2010; Vol 81:887-892.
2. Winner, John. Antibodies and clinical neurological syndromes. The Biomedical Scientist. Jul 2006; 622-624.

We help scientists achieve their goals in advanced toxicological testing by offering a wide range of preclinical, species-specific assays.

Toxicological side effects are a common obstacle in drug development pipelines. They often manifest in site-specific damage to vital organs, including the liver, kidney and heart. Labs engaged in compound, drug and device screening and development are chartered to perform a comprehensive survey of potential toxicological side effects as part of their routine practice.


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